- Scene 1—The Duchess's palace in Malfi: Antonio and Delio are discussing the former's return from France, and discussing how the French king runs his court, comparing it to an easily poisoned fountain. They are interrupted by the entry of Bosola and the Cardinal. Antonio and Delio hold their conversation, stepping to the background to watch as Bosola angrily tries to gain the Cardinal's pardon, speaking of the time he has spent in the galleys in penal servitude, and in the service of the Cardinal. Bosola declares that he is surely done with service, but the Cardinal is not interested in Bosola's new merit and takes his leave. Bosola compares himself to Tantalus, never able to acquire the thing he most desires, like an injured soldier who can only depend on his crutches for support of any kind. When he leaves, Antonio and Delio comment on his past offense, and how he will surely come to no good if he is kept in neglect. Ferdinand comes into the palace, talking to his courtiers about a tournament that Antonio has just won. When the Cardinal, Duchess, and Cariola enter to speak with Ferdinand, Antonio and Delio have a moment to themselves to discuss the Cardinal's character; he is found to be a very dishonest, disagreeable person, as is his brother, Ferdinand. Only their sister, the Duchess, earns the approval of everyone, a very pleasant and gracious woman. After the two gentlemen leave, Ferdinand petitions his sister to make Bosola the manager of her horses; when everyone else leaves, Ferdinand and the Cardinal reveal that it is because Bosola is to spy on their sister. When Bosola is brought in and made aware of this plan, he at first refuses, but ultimately is given no choice. The Cardinal and Ferdinand then turn their attention to their sister, urging her not to marry again, now that she is a widow, going so far as to threaten her with death, in Ferdinand's case. She refuses to be bullied, and once her brothers are out of sight, she proposes to Antonio by giving him her wedding ring. Having Cariola, the Duchess's maid, as their witness, this private ceremony is legally binding and the Duchess and Antonio become husband and wife.
- Scene 1—The Duchess's palace in Malfi, nine months later: Bosola and Castruchio enter, Bosola criticizing his companion's appearance, and telling him that he would make a ridiculous judge. When an old woman intrudes on their conversation, Bosola's insults turn on her, calling her hideous to the point that no amount of make-up would help. He also accuses her of being too like a witch; the old lady and Castruchio leave Bosola alone to muse on the mysterious way the Duchess is acting of late. He believes she is pregnant (no one but Delio and Cariola know that the Duchess and Antonio are married), and aims to prove it by using apricots both to spark her pregnant appetite and to induce labor, as apricots were believed to do. The Duchess, when she enters, accepts the fruit from Bosola, and quickly starts going into labor. She then retires to her chamber claiming to be ill, with a worried Antonio following in her wake.
- Scene 2—Same place and time as the previous scene: Bosola, alone, realizes that the Duchess is indeed pregnant. After accosting the hapless old lady again, he watches as Antonio and the servants in a commotion about a Swiss mercenary who had invaded the Duchess's room, and the loss of several jewels and gold utensils. Even with all the uproar, Antonio is not distracted from his wife's "illness"; she is actually in labor. Cariola, the lady's maid, enters with good news once Antonio is alone—he is the father of a son.
- Scene 3—Same place and time as the previous scene: Bosola re-enters the now empty room, having heard a woman (the Duchess) shriek. Antonio discovers him and questions his purpose in being there, since everyone had been commanded to keep to their rooms. Antonio tells him to stay away from the Duchess since he doesn't trust Bosola. In Antonio's agitation, he accidentally drops a horoscope for his son's birth, which Bosola retrieves. He realizes what it means, and resolves to send it to the Duchess's brothers with Castruccio.
- Scene 4—The Cardinal's rooms: The Cardinal and his mistress, Julia, are discussing their rendezvous when a messenger calls the Cardinal away with an important message. Delio enters to find Julia alone. He was once a suitor of hers and offers her money. Julia leaves to meet her husband, Castruccio, and Delio fears that her husband's arrival means Antonio's secret marriage is about to be revealed.
- Scene 5—Rome, in Ferdinand's private apartments: An enraged Ferdinand, with the letter from Bosola, and his brother the Cardinal, meet to discuss what they think is an awful treachery by their sister. Ferdinand is angry to the point of shouting about his sister's "whorish" behavior (he knows of the child, but not of the marriage), and the Cardinal struggles to control his brother's temperamental outburst. Ferdinand resolves to discover the man his sister is seeing, threatening all and sundry.
- Scene 1—The Duchess's palace in Malfi, after some time has passed: Antonio greets the returning Delio, who has come from Rome with Ferdinand. Antonio reveals that the Duchess has had two more children in the time Delio was gone. Antonio fears the wrath of the recently arrived Ferdinand, and Delio tells him the ordinary people think the Duchess is a whore. While they talk, the Duchess and Ferdinand enter. He tells her that he has found a husband for her, the Count Malateste. She disregards this, as she is already married (still secretly of course) to Antonio. When left alone, Ferdinand consults with Bosola to discover the father of the three seemingly illegitimate children; Bosola has acquired a skeleton key to the Duchess's room, which Ferdinand takes, telling him to guess what will happen next.
- Scene 2—The Duchess's bedchamber: Antonio comes up to the Duchess's bedroom to spend the night, and they banter back and forth about the point of lovers just sleeping together. Antonio and Cariola leave to allow the Duchess to complete her night-time preparations, but she is not alone; Ferdinand sneaks in and startles her. He gives her a knife, intending her to kill herself, and his fury increases when she tells him she is married without his knowledge. Ferdinand leaves, declaring he will never see her again. He exits just in time, for Antonio bursts in brandishing a pistol, but the Duchess forces him to leave again when Bosola knocks at the door. Bosola informs the Duchess that Ferdinand has left for Rome again, and she tells him that Ferdinand's bills of exchange (he has so far dealt with her accounts) will no longer work, since Antonio has been false with her accounts. This is, of course, a trick to get Antonio out of Malfi; she calls Antonio back in (once Bosola exits) to tell him to flee to Ancona, where she will send him all her treasure and valuables. The couple puts on a show argument for the benefit of the returning Bosola and officers, where she criticises his faulty record keeping and banishes him. Bosola does not believe the Duchess was justified in banishing Antonio, and tells her that Antonio is a good, honest man. This speech prompts the Duchess to confide the secret marriage to Bosola. He is then left on stage to lament his role as a spy, for now he must reveal all to Ferdinand.
- Scene 3—A room in a palace at Rome: The Cardinal, Ferdinand, Malateste, Pescara, Silvio and Delio are discussing the new fortifications that are being made in Naples. Ferdinand and his men, leaving the Cardinal and Malateste to speak privately, are very harsh in their critique of Malateste, considering him too cowardly to fight in an upcoming battle. Bosola, meanwhile, interrupts the Cardinal's private conference with news of his sister. The Cardinal leaves to petition for her and her family's exile from Ancona, while Bosola goes to tell the Duchess's first child (from her first husband) what has happened with his mother. Ferdinand goes to find Antonio.
- Scene 4—The shrine of Our Lady of Loreto, Italy, in the Ancona province: Two pilgrims are visiting the shrine in Ancona, and witness the Cardinal being symbolically prepared for war. The Cardinal then proceeds to take the Duchess's wedding ring, banish her, Antonio, and their children, while the pilgrims muse over the reason for what they have just seen.
- Scene 5—Near Loreto: The newly banished family, and the maid Cariola, enter Loreto. Shortly after their arrival, Bosola comes and presents the Duchess with a letter from Ferdinand, which indirectly states that Ferdinand wants Antonio dead. Antonio tells Bosola that he will not go to Ferdinand, and the Duchess urges him to take the oldest child and go to Milan to find safety, which he promptly does. Bosola and masked guards then take the Duchess and her remaining children captive, on the orders of her brothers.
- Scene 1—A prison (or the Duchess's lodgings serving as a prison) near Loreto: Ferdinand comes in with Bosola, who is describing to him how the Duchess is dealing with her imprisonment. It seems she is not affected to Ferdinand's satisfaction, and he leaves angrily. Bosola greets the Duchess, telling her that her brother wishes to speak with her, but will not do so where he can see her. She agrees to meet with her brother in the darkness. Once the lights are out, Ferdinand returns. He presents her with a dead man's hand, leading her to believe that it is Antonio's, with her wedding ring on it. He then exits, leaving Bosola to show the Duchess lifelike figures of her husband and children, made to appear as though her family was dead. The Duchess believes them to be the genuine articles, and resolves to die—her despair is so deep it affects Bosola. When she leaves, Ferdinand re-enters; Bosola pleads with him to send his sister to a convent, refusing to be a part of the plot any more. Ferdinand is beyond reason at this point, and tells Bosola to go to Milan to find the real Antonio.
- Scene 2—Same place and time as the previous scene: The Duchess and her maid, Cariola, come back, distracted by the noises being made by a group of madmen (Ferdinand brought them in to terrorise her). A servant tells her that they were brought for sport, and lets in several of the madmen. Bosola, too, sneaks in with them, disguised as an old man, and tells the Duchess that he is there to make her tomb. When she tries to pull rank on him, executioners with cords and a coffin come in. Cariola is removed from the room, leaving Bosola and the executioners with the Duchess. The Duchess makes a brave show, telling the executioners to "pull, and pull strongly", welcoming her strangulation. Cariola is brought back, and after struggling fiercely, she too is strangled. Ferdinand comes to view the scene, and is also shown the bodies of his sister's children, who were murdered as well. Ferdinand reveals that he and the Duchess were twins, and that he had hoped, if she had remained a widow, to inherit all her wealth. Bosola, sensing that Ferdinand is ready to turn on him next, demands payment for his atrocities. Ferdinand, distracted, leaves him alone with the bodies. Astonishingly, the Duchess is not dead. A shocked Bosola has no time to call for medicine; he manages to tell the Duchess that Antonio is not really dead; that the figures she saw were fake, before she finally dies. Bosola, remorseful at last, takes her body to the care of some good women, planning to leave immediately thereafter for Milan.
- Scene 1—Outside Ferdinand and the Cardinal's palace in Milan: Antonio returns to see if he can reconcile with Ferdinand and the Cardinal, but Delio is dubious as to the wisdom of this. Delio asks Pescara, a marquis, to give him possession of Antonio's estate for safekeeping, but Pescara denies him. Julia presents Pescara with a letter from the Cardinal, which states that she should receive Antonio's property, and which Pescara grants to her. When Delio confronts him about this, Pescara says that he would not give an innocent man a property that was taken from someone by such vile means (the Cardinal took the property for himself once Antonio was banished), for it will now become an appropriate place for the Cardinal's mistress. This statement impresses the hidden Antonio. When Pescara leaves to visit an ill Ferdinand, Antonio decides to pay a night-time visit to the Cardinal.
- Scene 2—Inside the same palace: Pescara, come to visit Ferdinand, is discussing his condition with the doctor, who believes Ferdinand may have lycanthropia: a condition whereby he believes he is a wolf. The doctor thinks there is a chance of a relapse, in which case Ferdinand's diseased behaviour would return; namely, digging up dead bodies at night. Pescara and the doctor make way for the mad Ferdinand, who attacks his own shadow. The Cardinal, who has entered with Ferdinand, manages to catch Bosola, who has been watching Ferdinand's ravings. The Cardinal assigns Bosola to seek out Antonio (by following Delio) and then slay him. After the Cardinal leaves, Bosola does not even make it to the door before he is stopped by Julia, who is brandishing a pistol. She accuses him of having given her a love potion, and threatens to kill him to end her love. Bosola manages to disarm her and convince her to gather intelligence for him about the Cardinal. Bosola then hides while Julia uses all of her persuasive powers to get the Cardinal to reveal his part in the death of his sister and her children. The Cardinal then makes Julia swear to keep silent, forcing her to kiss the poisoned cover of a bible, causing her to die almost instantly. Bosola comes out of hiding to confront the Cardinal, although he declares that he still intends to kill Antonio. Giving him a master key, the Cardinal takes his leave. However, once he is alone, Bosola swears to protect Antonio, and goes off to bury Julia's body.
- Scene 3—A courtyard outside the same palace: Delio and Antonio are near the Duchess's tomb; as they talk, an echo from the tomb mirrors their conversation. Delio leaves to find Antonio's eldest son, and Antonio leaves to escape the distressing echo of his wife's resting place.
- Scene 4—The Cardinal's apartments in Milan: The Cardinal enters, trying to dissuade Pescara, Malateste, Roderigo and Grisolan from staying to keep watch over Ferdinand. He goes so far as to say that he might feign mad fits to test their obedience; if they come to help, they will be in trouble. They unwillingly exit, and Bosola enters to find the Cardinal planning to have him killed. Antonio, unaware of Bosola, sneaks in while it is dark, planning to seek audience with the Cardinal. Not realising who has entered, Bosola attacks Antonio; he is horrified to see his mistake. He manages to relate the death of the Duchess and children to the dying Antonio, who is glad to be dying in sadness, now that life is pointless for him. Bosola then leaves to bring down the Cardinal.
- Scene 5—The same apartments, near Julia's lodging: The Cardinal, unaware of what has just happened, is reading a book when Bosola enter with a servant, who is bearing Antonio's body. He threatens the Cardinal, who calls for help. Help is not forthcoming, for the gentlemen from the beginning of the previous scene, while they can hear him calling, have no desire to go to his aid (because of his previous order to not at any cost try to help Ferdinand). Bosola kills the servant of the Cardinal first, and then stabs the Cardinal. Ferdinand bursts in, also attacking his brother; in the fight, he accidentally wounds Bosola. Bosola kills Ferdinand, and is left with the dying Cardinal. The gentlemen who heard the cries now enter the room to witness the deaths of the Cardinal and Bosola. Delio enters too late with Antonio's eldest son, and laments the unfortunate events that have passed.
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